Method of treating patients with a mucinous glycoprotein (MUC-1) vaccine

ABSTRACT

The present invention provides a method for treating an individual who is afflicted with a cancer, such as non-small cell lung cancer or prostate cancer, by administering to that individual a MUC-1-based formulation. The formulation may be a MUC-1 based liposomal vaccine formulation.

CROSS-REFERENCE

This non-provisional application claims priority to U.S. patentapplication Ser. No. 11/475,250, filed Jun. 27, 2006, which claims thebenefit of U.S. Provisional Application Ser. No. 60/694,233, filed onJun. 28, 2005, all of which are incorporated herein in their entiretiesby reference.

FIELD OF THE INVENTION

The present invention relates to a method for identifying individualswith cancer that are suitable for treatment with a mucinous-glycoprotein(MUC-1)-based formulation. Examples of such cancers are non-small celllung cancer and prostate cancer. In some cases, the MUC-1-basedformulation is a BLP25 vaccine.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of cancer-related mortality for bothsexes in North America. In 2004, approximately 174,000 new cases of lungcancer (54% in men, 46% in women) were diagnosed in the U.S. Moreover,in 2004 approximately 160,000 people died of this disease in the U.S.alone.

Unfortunately, at the time of diagnosis, only 25% of lung cancerpatients are potentially curable by surgery. Furthermore, chemotherapyhas only modestly improved the chances of survival in individualsafflicted with the cancer.

Non-small cell lung cancer (NSCLC) is the most common of lung cancers,as it accounts for approximately 75 to 80% of all primary lung cancers.NSCLC is typified by squamous cell carcinoma, adenocarcinoma, and largecell carcinoma. It has been observed that the mucinous glycoprotein,MUC-1, is highly expressed in such carcinomas, beyond levels of normalexpression in epithelial cells of healthy individuals. It has also beenobserved that many carbohydrate moieties that adorn the MUC-1 proteinare shorter than those moieties attached to MUC-1 proteins of normalcells, by virtue of attachment to the MUC-1 polypeptide backbone. Thus,the MUC-1 polypeptide backbone in cancer cells is more exposed than thepolypeptide backbone in normal cells.

After lung cancer, prostate cancer is the second most common diagnosedcancer in men in the United States. Roughly 190,000 men are diagnosedwith prostate cancer in the United States and nearly 30,000 men die fromthe disease yearly.

Biochemical failure after prostatectomy (PR) for treatment of prostatecancer is often a harbinger of clinical failure, which may shorten thelife expectancy of the patient. And although a need exists foradditional non-invasive methods of treating prostate cancer, a specialneed exists for a treatment of men with post-prostatectomy biochemicalfailure.

There is a need in the art for identifying patients suitable for novelcancer therapies, as well as the development of such novel cancertherapies. The present invention provides a method for identifyingindividuals with cancer that are suitable for treatment with amucinous-glycoprotein (MUC-1)-based formulation.

SUMMARY OF THE INVENTION

The present invention relates to the identification and treatment ofindividuals with cancer, wherein the cancer is suitable for treatmentwith a MUC-1-based formulation. Examples of such cancers are NSCLC andprostate cancer. The present invention also encompasses the treatment ofother cancers in addition to NSCLC and prostate cancer with thedescribed MUC-1-based formulations.

In one embodiment of the invention, the MUC-1-based formulation may be aMUC-1-based liposomal vaccine. For instance, the liposomal vaccine maycomprise a MUC-1 peptide in its lipid bilayer or encapsulated within itsvesicle structure. The MUC-1 peptide also may be lipidated to facilitateits association with the liposomal lipid bilayer or membrane. The MUC-1peptide may comprise the amino acid sequence depicted in SEQ ID NO. 1 oran immunologically active fragment or variant thereof (collectivelyreferred to as a “functional variant”), or SEQ ID NO. 2, or animmunologically active fragment or variant thereof. Particularcharacteristics of MUC-1 core repeat variants are described below.

In another aspect of the present invention, a method (“Method 1”) isprovided for treating a subject with NSCLC or prostate cancer. Themethod comprises: (A) selecting for treatment a subject who has NSCLC orprostate cancer, and (B) administering to that subject, for a period oftime, a MUC-1-based formulation. In one embodiment of Method 1, theMUC-1 based formulation comprises a liposome that contains at least onepolypeptide having the amino acid sequence depicted in SEQ ID NO. 1 oran immunologically active fragment or variant thereof, or SEQ ID NO. 2,or an immunologically active fragment or variant thereof.

In specific embodiments, Method 1 may further include a step (C)comprising evaluating the treated subject. In individual embodiments,evaluating the treated subject may be accomplished by measuring animmune reaction in the treated subject. In certain embodiments,measuring the immune reaction in the treated subject can comprisemeasuring a T-cell proliferation. In yet other embodiments, evaluatingthe treated subject can comprise determining at least one or more of:(a) tumor size, (b) tumor location, (c) nodal stage, (d) growth rate ofthe NSCLC or prostate cancer, (e) survival rate of the subject, (f)changes in the subject's lung cancer or prostate cancer symptoms, (g)changes in the subject's PSA concentration, (h) changes in the subject'sPSA concentration doubling rate, (i) changes in the subject's quality oflife, or (j) a combination thereof.

In these embodiments, evaluating the subject may be performed before,during, or after the period of time. Evaluating the subject may also beperformed before and after the period of time.

In a further embodiment, the formulation is a BLP25 liposome vaccine.“BLP25” is a specific lipidated MUC-1 core repeat, identified below. TheBLP25 vaccine may comprise preformed liposomes that comprise a MUC-1core repeat, such as those depicted in SEQ ID NOs: 1 and 2. Thepreformed liposomes comprising a MUC-1 core repeat may be lyophilized.

In one embodiment of this method, the BLP25 liposome vaccine is in a kitand instructions for preparing and using the vaccine are included in thekit. Hence, the kit may comprise another liquid, such as a sodiumchloride solution (0.9%, USP) that can be used to reconstitute thatlyophilized material. Alternatively, the BLP25 liposome vaccine may besupplied as a liquid. The kit also may comprise an adjuvant, or acombination of adjuvants. Examples of adjuvants include, but are notlimited to, lipid A, muramyl dipeptide, alum, or a cytokine. Thus, thekit may comprise a number of vials or vessels that enable a person toprepare the BLP25 vaccine for administration.

The step of administering the formulation to the subject may be by anysuitable method, and utilizing any pharmaceutically acceptable dosageform. Examples of administration methods include, but are not limitedto, injection, wherein the injection is an intramuscular injection, asubcutaneous injection, intravenous, intranodal, intratumoral,intraperitoneal, or an intradermal injection. Alternatively, the vaccineor the liposomally-bound MUC-1 core repeat peptide may be administeredby aerosol, nasal, oral, vaginal, rectal, ocular, local (powders,ointments or drops), buccal, intracisternal, intraperitoneal, or topicaladministration, and the like. The vaccine or the liposomally-bound MUC-1core repeat peptide also may be administered via a formulation suitablefor transdermal delivery, such as via a transdermal patch.

In yet another aspect of the present invention, described is a method(“Method 2”) for improving or maintaining the quality of life of anindividual with cancer, such as NSCLC or prostate cancer. This methodmay comprise administering to a subject diagnosed with a cancersusceptible to treatment with a MUC-1 based formulation, such as NSCLCor prostate cancer, a dose of BLP25 liposome vaccine routinely for aperiod of time. In a further aspect of Method 2, a combined score of theindividual's physical well-being, functional well-being, and cancersymptoms before, during, and after a period of time may be calculated.

In one embodiment, the dose of BLP25 liposome vaccine provides about1,000 μg of the BLP25 MUC-1 lipopeptide, in one or multipleadministrations, although other doses, described below, may beadministered. See, for instance, the doses envisioned under the BLP25Dosages subsection below.

Another aspect of the present invention is a method for identifyingsubjects suitable for treatment with a MUC-1-based composition, such asa BLP25 vaccine. One embodiment of the method entails determiningwhether the level of circulating MUC-1 peptide in a subject'sbloodstream, or in a blood, plasma, urine, serum, or other suitablebiological sample, is normal or abnormal. If the level of circulatingMUC-1 is normal, then the subject is suitable for subsequent treatmentwith a MUC-1 composition. That is, a subject having a normal level ofcirculating MUC-1 can be given a dose of a MUC-1 vaccine, such as BLP25.In one embodiment, the upper limit of the normal level of circulatingMUC-1 is about 37.7 U/ml. Accordingly, a subject having a circulatingMUC-1 level of 37.7 U/ml or less is suitable for treatment with a MUC-1composition, such as a BLP25 vaccine. However, it is well within thepurview of the skilled person to determine threshold levels ofcirculating MUC-1 peptides in different groups of individuals. That is,the value “37.7 U/ml” is not necessarily a definitive threshold for allassayable populations. A “normal” quantity of circulating antigen can bedetermined from a desired subpopulation and used as an indicator toclassify MUC-1 quantities as either normal or abnormal.

In another embodiment, the method entails detecting the presence of HLAA2 protein or encoding nucleic acid or an HLA A2 RNA transcript in asubject's bloodstream, or in a blood, plasma, urine, serum, or othersuitable biological sample. Detection of the presence of HLA A2 proteinor encoding nucleic acid or an HLA A2 RNA transcript in the biologicalsample indicates that the subject is suitable for treatment with a MUC-1based composition, such as a BLP25 vaccine.

In one embodiment, the subject screened for suitability for treatmentwith a MUC-1 composition has stage IIIB or stage IV non-small cell lungcancer.

In one aspect of the present invention is a method for treating asubject with cancer. This method comprises (a) selecting for treatment asubject having a cancer cell that expresses MUC-1, and (b) administeringto that subject, for a period of time, a MUC-1-based formulation,wherein the formulation comprises: (i) a liposome; and (ii) at least onepolypeptide comprising the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO. 1, animmunologically active variant of the amino acid sequence of SEQ ID NO.1, the amino acid sequence of SEQ ID NO. 2, and an immunologicallyactive variant of the amino acid sequence of SEQ ID NO. 2, and whereinthe subject does not have a high level of circulating MUC-1 in thesubject's serum. In one embodiment, the cancer is selected from thegroup consisting of ovarian cancer, liver cancer, colon cancer, breastcancer, pancreatic cancer, kidney cancer, head and neck cancer, andmultiple myeloma.

Both the foregoing general description and the following briefdescription of the drawings are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following detailed description ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the results from a study, detailed herein,showing the overall survival by study arm between patients receivingtreatment with BLP25 liposomal vaccine or patients receiving only bestsupportive care (BSC). See example 1 below.

FIG. 2 is a graph demonstrating survival analysis for stage IIIBlocoregional patients with NSCLC. The survival analysis for the twogroups of patients (treatment and BSC) includes the survivaldistribution function of patients treated with BLP25 liposomal vaccineversus patients treated only with best supportive care. See example 1below.

FIG. 3 is a graph depicting the percentage change in prostate-specificantigen (“PSA”) doubling time for different patients who had received adose of BLP25 liposomal vaccine. See example 3 below.

FIG. 4 is a table listing the frequency distribution of patient CA27.29values by visit, arm (BLP25 or Control), and normal/abnormal level.

FIG. 5 displays the survival curve for patients with normal levels ofthe antigen at baseline compared to those patients who had abnormallevels.

FIG. 6 shows median survival of those patients with normal levels ofCA27.29 was 24.2 months while that of patients with abnormal levels was9.8 months (Cox p=0.0006).

FIG. 7 shows data from the control arm where the median survival ofpatients with normal levels of CA27.29 was 15.1 months as compared to11.3 months for those with abnormal CA27.29 levels (Cox p=0.0042).

FIG. 8 shows data of patients with pre-existing normal levels of CA27.29in the BLP25 arm had a median survival of 24.2 months as compared tothose patients in the Control arm who had a median survival of 15.1months (Cox p=0.0605).

FIG. 9 shows that the patients with pre-existing abnormal levels ofCA27.29 in the BLP25 arm had a median survival of 9.8 months and thosepatients on the control arm had a median survival of 11.3 months (Coxp=0.5234).

FIG. 10 shows the Kaplan-Meier Curves of survival duration by T cellproliferation.

FIG. 11 illustrates the survival analyses that were performed for thechosen HLA types between treatment arms.

FIG. 12 shows the survival of patients with HLA A02 in each of the studyarms.

FIG. 13 demonstrates the differences in survival between the two arms inpatients with the DQB 1-05 allele.

FIG. 14 displays the survival of patients in both arms who have theDRB1-04 haplotype.

FIG. 15 shows another survival curve for patients in both arms who havethe DQB 1-02 allele.

FIG. 16 illustrates additional survival analyses within the treatmentarms for the same haplotypes listed in FIG. 11.

FIG. 17 shows Kaplan-Meier Survival curves for patients in the BLP25 armonly who have the CW07 allele (n=45) versus those patients who do nothave the allele (n=43).

FIG. 18 shows survival by treatment arm of patients with stage IIIBmalignant pleural effusion or stage IV disease at study entry.

FIG. 19 shows survival in terms of baseline CA27.29 normal vs. abnormal(control arm).

FIG. 20 shows overall survival baseline CA27.29 normal vs. abnormal.

FIG. 21 shows overall survival by treatment arm.

FIG. 22 shows survival by treatment arm of patients with stage IIIBlocoregional disease at study entry.

FIG. 23 survival in terms of baseline CA27.29 normal vs. abnormal (BLParm).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides MUC-1 based formulations and methods fortreating an individual who is afflicted with cancer, wherein the canceris susceptible to treatment with a MUC-1 formulation. Examples of suchcancers are NSCLC and prostate cancer. The present invention alsoencompasses the treatment of other cancers in addition to NSCLC andprostate cancer with the described MUC-1-based formulations.

According to the present invention, a formulation may comprise a MUC-1core repeat. A MUC-1 core repeat may be a sequence of amino acids thatoccurs any number of times in a MUC-1 protein. Preferably, a MUC-1 corerepeat peptide of the present invention mimics the exposed nature of aMUC-1 protein expressed in cancer cells, which have shorter carbohydratemoieties attached to the MUC-1 protein backbone.

In one embodiment, a MUC-1 core repeat of the present invention has theamino acid sequence, STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID NO. 1).

A MUC-1 core repeat may also have the amino acid sequence depicted inany of the following:

(SEQ ID NO: 2) STAPPAHGVTSAPDTRPAPGSTAPPK(palmitoyl)G (SEQ ID NO: 3)STAPPAHGVTSAPDTRPAPG 

In certain embodiments, this core repeat may be lipidated. One suchMUC-1 core repeat lipopeptide is referred to herein as BLP25. Theformulation may also be associated with a liposome. This association mayinclude, but is not limited to, incorporation of the peptide into theliposome or encapsulation of the peptide by the liposome.

A liposome vaccine that contains a BLP25 lipopeptide is referred toherein as “L-BLP25.”

The formulations of the invention may further comprise an adjuvant, or acombination of adjuvants, such as lipid A or interleukin-2 (IL-2). Otherexemplary adjuvants useful in the invention are described below. TheMUC-1-based formulation may be formulated as a vaccine, and the vaccinemay be a liposomally-associated MUC-1 core repeat vaccine. In severalembodiments, the vaccine formulation comprises a liposomally-associatedMUC-1 core repeat and an adjuvant. The MUC-1 core repeat can belipidated.

A vaccine of the present invention may comprise: (a) a MUC-1 core repeatcomprising the sequence of SEQ ID NO.: 1 and exogenous lipid; or (b) aMUC-1 core repeat comprising the sequence of SEQ ID NO.: 1 and aliposome; or (c) a MUC-1 core repeat comprising the sequence of SEQ IDNO.: 1 and a liposome and an adjuvant; or (d) a MUC-1 core repeatcomprising the sequence of SEQ ID NO.: 1 and a liposome and an adjuvant,where the adjuvant is lipid A.

In certain other embodiments, a vaccine of the present invention maycomprise (a) a MUC-1 core repeat comprising the sequence of SEQ ID NO.:2 and exogenous lipid; or (b) a MUC-1 core repeat comprising thesequence of SEQ ID NO.: 2 and a liposome; or (c) a MUC-1 core repeatcomprising the sequence of SEQ ID NO.: 2 and a liposome and an adjuvant;or (d) a MUC-1 core repeat comprising the sequence of SEQ ID NO.: 2 anda liposome and an adjuvant, where the adjuvant is lipid A.

The concept of treating individuals having a cancer susceptible totreatment with a MUC-1-based formulation, such as NSCLC or prostatecancer, as well as the constituents of the MUC-1-based vaccine formula,are described in more detail below.

I. BLP25 Liposome Vaccine

In one embodiment, the MUC-1-based formulation comprises a certainamount of MUC-1 lipopeptide BLP25 and a certain amount of adjuvant. Sucha formulation is referred to herein as a BLP25 Liposome Vaccine(“L-BLP25”), which may be in a liquid or lyophilized formulation. Forinstance, the formulation or vaccine may contain, in a single dosageamount, about 1000 μg of MUC-1 lipopeptide BLP25 and about 500 μg oflipid A.

Other microgram amounts of MUC-1 lipopeptide and lipid A, however, areenvisioned in this invention. For instance, the amount of BLP25lipopeptide may be sufficient to accommodate multiple doses of thevaccine. Hence, the MUC-1 core repeat formulation may contain, forexample, about 50 μg, about 100 μg, about 200 μg, about 300 μg, about400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about900 μg, about 1,000 μg, about 1,010 μg, about 1,020 μg, about 1,030 μg,about 1,040 μg, about 1,050 μg, about 1,060 μg, about 1,070 μg, about1,080 μg, about 1,090 μg, about 1,100 μg, about 1,200 μg, about 1,300μg, about 1,400 μg, about 1,500 μg, about 1,600 μg, about 1,700 μg,about 1,800 μg, about 1,900 μg, about 2,000 μg, about 3000 μg, about4000 μg, about 5000 μg, about 6000 μg, about 7000 μg, about 8000 μg,about 9000 μg, about 10000 μg, about 15000 μg, about 25000 μg, or moreof MUC-1 core repeat. One particular dosage of MUC-1 core repeat is inthe range of about 500 μg to about 1500 μg, about 500 μg to about 1500μg, and about 1000 μg.

Similarly, the amount of lipid A may be varied to match the amount ofMUC-1 peptide formulated into the vaccine. Hence, the amount of lipid Amay be about 50 μg, about 100 μg, about 200 μg, about 300 μg, about 400μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900μg, about 1,000 μg, about 1,010 μg, about 1,020 μg, about 1,030 μg,about 1,040 μg, about 1,050 μg, about 1,060 μg, about 1,070 μg, about1,080 μg, about 1,090 μg, about 1,100 μg, 1,200 μg, 1,300 μg, 1,400 μg,1,500 μg, 1,600 μg, 1,700 μg, 1,800 μg, 1,900 μg, or about 2,000 μg, ormore. In particular there may be about 500 μg of lipid A.

The BLP25 lipopeptide and lipid A may be associated with the lipidbilayer of the liposomes that are formed upon rehydration of the drypowder.

The formulation may be retained in a vial, such as in a 5 ml Type Iborosilicate glass vial. The vial, which contains the MUC-1 formulation,also may contain other vaccine ingredients. For instance, the vial maycomprise additional liposomal lipids, such as dipalmitoylphosphatidiylcholine, cholesterol, and dimyristoyl phosphatidylglycerol.Each amount of those particular lipids may vary. Therefore, the amountof any one of dipalmitoyl phosphatidiylcholine, cholesterol, anddimyristoyl phosphatidylglycerol in a vial may be about 1 mg, about 2mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg,about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg,about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg,or about 100 mg, or more than about 100 mg. The liposomal lipids may becontained in a second vial to the vial while the MUC-1 formulation maybe contained in a first vial.

Other embodiments, of course, exist for the purposes of this invention.Hence, the above amounts of MUC-1 lipopeptide BLP25, adjuvant, andliposomal lipids in the L-BLP25 are given as examples only. Determiningthe appropriate amount of each constituent, including amounts of MUC-1lipopeptide, can be readily accomplished. In some embodiments, theamount of MUC-1 lipopeptide will be greater or less than about 300 μg.The vaccine does not need to be supplied in a 5 ml Type I borosilicateglass vial, but may be supplied in any manner known in the art.

In one embodiment, the BLP25 lipopeptide is a linear 27-residue peptidethat contains a lipidated amino acid derivative close to its C-terminus.Specifically, BLP25 comprises a palmitoyl lipid on a lysine residue atposition 26 of the polypeptide. The sequence of the BLP25 lipopeptide isdepicted in SEQ ID NO.: 2, shown below:

SEQ ID NO. 2: STAPPAHGVTSAPDTRPAPGSTAPPK(palmitoyl)G

In other embodiments possessing a MUC-1 core sequence, an amino acid,such as threonine, serine, lysine, arginine, or cysteine, which mayoccur within the natural sequence of the peptide, may be a convenientsite to which a lipid can be linked. In addition, the lipid can belinked to a synthetic amino acid or an amino acid not naturally found inthe MUC-1 core sequence. Moreover, one or more of either the natural orsynthetic amino acids can be added to either end or within the MUC-1core sequence to facilitate the linking of a lipid.

The number of amino acids that can be added to the MUC-1 core sequenceis not meant to be limiting, as any number of amino acids can be addedas long as the peptide still functions in the methods of the invention.As demonstrated above, two additional amino acids have been added to theBLP25 polypeptide. That is, the C-terminus of the MUC-1 core sequenceends with a proline and is, therefore, 25-residues in length. In thecase of the BLP25 polypeptide, however, a lysine and a glycine have beenadded to that C-terminal proline to facilitate the linkage of palmitoyl.Hence, the length of the BLP25 polypeptide is 27 amino acids long.Conventional peptide synthesis methods can be used to add one or more ofsuch additional amino acids to a peptide sequence. Alternatively, theMUC-1 core sequence peptide or BLP25 can be made recombinantly.

In one particular embodiment, a BLP25 Liposome Vaccine (“L-BLP25”) maycomprise BLP25 lipopeptide, lipid A, cholesterol, DMPG, and DPPC. TheBLP25 lipopeptide may comprise the sequence of SEQ ID NO: 2, animmunologically active fragment, or an immunologically active variantthereof. A dose of such a BLP25 Liposome Vaccine may comprise about 1000μg of BLP25 lipopeptide, about 500 μg of lipid A, about 17.3 mg ofcholesterol, about 3.6 mg of DMPG, and about 29.1 mg of DPPC.

This particular vaccine composition and dosage also can be described in“per vial” amounts. Hence, a vial may comprise about 300 μg of BLP25lipopeptide, about 150 μg of lipid A, about 5.2 mg of cholesterol, about1.1 mg of DMPG, and about 8.7 mg of DPPC.

This vaccine may be lyophilized and then reconstituted prior toadministration, such as in sodium chloride solution. The BLP25 LiposomeVaccine quantities described above may be reconstituted, for example, inabout 0.6 ml of liquid, although any volume of liquid, depending on thedosage desired, may be used, such as about 0.1 ml, 0.2 ml, 0.3 ml, 0.4ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 10 ml, 11 ml, 12 ml, 13 ml,14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, or 20 ml, or more than 20 ml.The volume of liquid into which a lyophilized MUC-1 vaccine isreconstituted is not necessarily the volume that is administered to anindividual.

A. MUC-1 Core Repeat Variants

As an alternative to the MUC-1 core repeat sequence depicted in SEQ IDNOs: 1 and 2, the formulation of the invention may incorporateimmunologically active homologues or variants of those MUC-1 corerepeats. Accordingly, the present invention encompasses the use of aMUC-1 core repeat peptide having a sequence that is similar to, but notidentical to, the amino acid sequence depicted in either SEQ ID NO: 1 orSEQ ID NO: 2. Thus, the present invention contemplates the use of aMUC-1 core repeat that has a sequence identity of 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,81%, or 80% compared to the sequence of SEQ ID NO. 1 or SEQ ID NO: 2,and which is immunologically active.

A MUC-1 core repeat protein of the present invention may be modified tocontain conservative variations or may be modified so as to changenon-critical residues or residues in non-critical regions. Amino acidsthat are not critical can be identified by methods known in the art,such as site-directed mutagenesis, crystallization, nuclear magneticresonance, photoaffinity labeling, or alanine-scanning mutagenesis(Cunningham et al., Science, 244:1081-1085 (1989); Smith et al., J. Mol.Biol., 224:899-904 (1992); de Vos et al., Science, 255:306-312 (1992)).Modified proteins can be readily tested for activity or ability toinduce an immune response via methods such as protease binding tosubstrate, cleavage, in vitro activity, or in vivo activity.

Specifically, a MUC-1 core repeat variant may incorporate 1, 2, 3, 4, or5 amino acid substitutions that improve MUC-1 core repeat stability orwith a different hydrophobic amino acid that improves MUC-1 core repeatstability against oxidation, or with a different amino acid thatimproves MUC-1 core repeat stability against protease. Thus, a “variant”MUC-1 core repeat polypeptide of the invention can differ in amino acidsequence from the sequence represented in SEQ ID NOs: 1 or 2 by one ormore substitutions, deletions, insertions, inversions, truncations, or acombination thereof. Such a variant can be made to contain amino acidsubstitutions that substitute a given amino acid with another amino acidof similar characteristics. Conservative substitutions include, amongthe aliphatic amino acids interchange of alanine, valine, leucine, andisoleucine; interchange of the hydroxyl residues serine and threonine,exchange of the acidic residues aspartate and glutamate, substitutionbetween the amide residues asparagine and glutamine, exchange of thebasic residues lysine and arginine, and replacements among the aromaticresidues phenylalanine and tyrosine. See Bowie et al., Science,247:1306-1310 (1990).

B. MUC-1 Core Repeat Fusion Proteins

A MUC-1 core repeat peptide having the full-length sequence of SEQ IDNOs: 1 or 2, or a variant thereof, can also be joined to anotherpolypeptide with which it is not normally associated. Thus, a MUC-1 corerepeat peptide can be operatively linked, at either its N-terminus orC-terminus, to a heterologous polypeptide having an amino acid sequencenot substantially homologous to the MUC-1 core repeat. “Operativelylinked” indicates that the MUC-1 core repeat peptide and theheterologous polypeptide are both in-frame.

A fusion protein may, or may not, affect the ability of the MUC-1 corerepeat, or a functional variant thereof, to induce an immunologicalreaction from a host system. For example, the fusion protein can be aGlutathione S-transferase (GST)-fusion protein in which MUC-1 corerepeat is fused to the C-terminus of the GST sequence or an influenza HAmarker. Other types of fusion proteins include, but are not limited to,enzymatic fusion proteins, for example, beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly-His fusions, and Ig fusions. Suchfusion proteins, particularly poly-His fusions, can facilitate thepurification of recombinantly-produced MUC-1 core repeat for use in theinvention. In certain host cells, expression and/or secretion of aprotein can be increased by using a heterologous signal sequence fusedto a protease that transports the MUC-1 core repeat peptide to anextracellular matrix or localizes the MUC-1 core repeat protein in thecell membrane.

Other fusion proteins may affect the ability of a MUC-1 core repeat toinduce an immunological reaction. For example, a subregion of a MUC-1core repeat can be replaced, for example, with the corresponding domainor subregion from another region of a MUC-1 protein. Accordingly,chimeric MUC-1 core repeats can be produced. Likewise, the affinity forsubstrate can be altered or even proteolysis of the substrate prevented.Accordingly, one may use a protein having a sequence of, for instance,SEQ ID NO: 1 or 2, or variant thereof, as a competitive inhibitor ofanother MUC-1 core repeat peptide.

C. MUC-1 Core Repeat Modifications

MUC-1 core repeat variants also encompass derivatives or analogs inwhich (i) an amino acid is substituted with an amino acid residue thatis not one encoded by the genetic code, (ii) the mature polypeptide isfused with another compound such as polyethylene glycol, or (iii)additional amino acids are fused to the MUC-1 polypeptide, such as aleader or secretory sequence or a sequence for purification of thepolypeptide.

Typical modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Particularly common peptide modifications that can be applied to MUC-1core repeat include glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation, andADP-ribosylation. See T. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed. (W. H. Freeman and Company, New York (1993)); Wold,F., Posttranslational Covalent Modification of Proteins, B. C. Johnson,Ed. (Academic Press, New York 1-12 (1983)); Seifter et al., Meth.Enzymol., 182: 626-646 (1990); and Rattan et al., Ann. N.Y. Acad. Sci.,663:48-62 (1992).

Modifications can be made anywhere in a MUC-1 core repeat polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common innaturally-occurring and synthetic polypeptides.

II. BLP25 Dosages

When a MUC-1-based formulation, including a MUC-1 core peptide, BLP25polypeptide, or BLP25 liposomal vaccine is being given to a subject, oneof skill in the art understands that the dosage may depend on severalfactors, including, but not limited to, the subject's weight, tumorsize, or tumor progression. Generally, as used herein, a subject thatreceives the MUC-1-based formulation is a single organism. In certainembodiments, a subject will be a mammal. Specifically, a subject may behuman, including being a male or a female. In many embodiments, thesubject will be a patient, or an individual awaiting or under medicalcare and treatment.

A subject may be given a dose of about 50 μg, about 100 μg, about 200μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700μg, about 800 μg, about 900 μg, about 1,000 μg, about 1,010 μg, about1,020 μg, about 1,030 μg, about 1,040 μg, about 1,050 μg, about 1,060μg, about 1,070 μg, about 1,080 μg, about 1,090 μg, about 1,100 μg,1,200 μg, 1,300 μg, 1,400 μg, 1,500 μg, 1,600 μg, 1,700 μg, 1,800 μg,1,900 μg, or about 2,000 μg of BLP25 MUC-1 polypeptide that is in theBLP25 liposome vaccine, in either single or cumulative applications. Inspecific embodiments, the dose given to the subject is about 1,000 μs ofthe MUC-1-based formulation per week.

A subject may receive a dose of the MUC-1-based formulation, forexample, multiple times daily, every day, every other day, once a week,or any other suitable dosing regimen. In one embodiment, routinelyadministering encompasses administering a dose of BLP25 liposome vaccineonce a week for a period of time. Of course, the dosing regimen maycomprise other permutations of MUC-1 peptide delivery. That is, thevaccine may be administered once, twice, three times, four times, fivetimes, six times, or more times a week. In some embodiments, subjectswill be given at least 5 doses over a period of time. In otherembodiments, subjects will be given greater than or fewer than 5 doses.Thus, a subject may receive a dose of about 1,000 μg of the MUC-1lipidated polypeptide every week. Alternatively, the subject may receivetwo doses of 500 μg, twice a week, or a daily 100 μg dose over fivedays.

These dosage examples are not limiting and only used to exemplifyparticular dosing regimens for administering about 1,000 μg of the MUC-1lipidated polypeptide. For instance, if the appropriate dose for a givensituation is 1,000 μg per week, the doses may be broken down into anynumber of permutations. This also holds true if the appropriate dose fora particular situation is greater than or less than 1,000 μg.

The period of time that the MUC-1-based formulation is administered tothe subject may be any suitable period. Examples of such suitableperiods include, but are not limited to, at least about 3 months, atleast about 4 months, at least about 5 months, at least about 6 months,at least about 7 months, at least about 8 months, at least about 9months, at least about 10 months, at least about 11 months, at leastabout 12 months, at least about 13 months, at least about 14 months, atleast about 15 months, at least about 16 months, at least about 17months, at least about 18 months, at least about 19 months, at leastabout 20 months, at least about 21 months, at least about 22 months, atleast about 23 months, or at least about 24 months or longer. Thetreatment period also may continue for longer than 24 months, ifdesired, such as for 30 months, 31 months, 32 months, 33 months, 34months, 35 months, 36 months, or longer than 36 months.

In another embodiment of the invention, the period of time of dosing forany of the methods described herein is for at least about 2 weeks, atleast about 4 weeks, at least about 8 weeks, at least about 16 weeks, atleast about 17 weeks, at least about 18 weeks, at least about 19 weeks,at least about 20 weeks, at least about 24 weeks, at least about 28weeks, at least about 32 weeks, at least about 36 weeks, at least about40 weeks, at least about 44 weeks, at least about 48 weeks, at leastabout 52 weeks, at least about 60 weeks, at least about 68 weeks, atleast about 72 weeks, at least about 80 weeks, at least about 88 weeks,at least about 96 weeks, or at least about 104 weeks.

The MUC-1-based formulation may be administered in different phases oftreatment. For example, the MUC-1-based formulation may be administeredin both a treatment phase and a maintenance phase. In some embodiments,the treatment phase will comprise administration of the MUC-1-basedformulation in weekly dosages, whereas the maintenance phase may be inlonger time periods, such as about every 6 weeks, about every 7 weeks,about every 8 weeks, about every 9 weeks, about every 10 weeks, aboutevery 11 weeks, about every 12 weeks, or longer. In some cases, thedosage given in the treatment phase will be greater than the dosagegiven in the maintenance phase. However, treatment and maintenancephases may be designed to a particular individual so the time anddosages between the treatment and maintenance phases may significantlyvary from the above examples. Generally, the maintenance phase may beginat any time deemed appropriate. For example, in some embodiments, thetreatment phase will be eight weeks and the maintenance phase willcontinue throughout the individual's lifetime. In other embodiments,only a treatment or a maintenance phase will be undertaken.

In yet further embodiments, the MUC-1-based formulation will be givenprophylacticly. In these embodiments, the administration of theMUC-1-based formulation may prevent an individual from developingcancer, such as NSCLC or prostate cancer. When the MUC-1-basedformulation is used prophylacticly, the dosage amount and regime can bereadily determined.

Physicians can determine the amount of time that a subject should remainon the MUC-1-based formulation. In some cases, it may be advantageous toadminister the MUC-1-based formulation for the rest of an individual'slifetime.

III. Liposomes

In many embodiments, the MUC-1-based formulation will be used withliposomes. Liposomes are microscopic vesicles that consist of one ormore lipid bilayers surrounding aqueous compartments. See e.g.,Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis. 12(Suppl. 1): S61 (1993), and Kim, Drugs, 46: 618 (1993). Becauseliposomes can be formulated with bulk lipid molecules that are alsofound in natural cellular membranes, liposomes generally can beadministered safely and are biodegradable. Thus, liposomes are oftenused in drug delivery.

Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and can vary in size with diameters ranging from about0.02 μm to greater than about 10 μm. A variety of agents can beencapsulated in or inserted into liposomes. Hydrophobic agents partitionin the bilayers and hydrophilic agents partition within the inneraqueous space(s). See e.g., Machy et al., LIPOSOMES IN CELL BIOLOGY ANDPHARMACOLOGY (John Libbey, 1987), and Ostro et al., American J. Hosp.Pharm. 46: 1576 (1989).

Liposomes can adsorb to virtually any type of cell and then release anincorporated agent. In some cases, the liposome can fuse with the targetcell, whereby the contents of the liposome then empties into the targetcell. Alternatively, a liposome may be endocytosed by cells that arephagocytic. Endocytosis is followed by intralysosomal degradation ofliposomal lipids and release of the encapsulated agents. Scherphof etal., Ann. N.Y. Acad. Sci., 446: 368 (1985).

Additionally, liposomes can be used to present active agents, such aspolypeptides, at their surface and, therefore, induce various events,such as signaling cascades or initiate biochemical pathways, withoutfusing to a target cell or surface as mentioned in the precedingparagraph. Thus, for example, a polypeptide may be incorporated into thelipid bilayer, for instance, of a liposome via a lipid that is attachedto the polypeptide.

Liposomes are used as delivery vehicles with the MUC-1-basedformulations of the present invention. Exemplary suitable liposomes thatmay be used in the methods of the invention include multilamellarvesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV),small unilamellar vesicles (SUV), medium-sized unilamellar vesicles(MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles(GUV), multivesicular vesicles (MVV), single or oligolamellar vesiclesmade by reverse-phase evaporation method (REV), multilamellar vesiclesmade by the reverse-phase evaporation method (MLV-REV), stableplurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesiclesprepared by extrusion methods (VET), vesicles prepared by French press(FPV), vesicles prepared by fusion (FUV), dehydration-rehydrationvesicles (DRV), and bubblesomes (BSV). However, as understood by onehaving skill in the art, the type of liposome is not meant to belimiting and may include any liposome made in any matter that iscompatible with the methods of the invention. Techniques for preparingliposomes are well known in the art. See COLLOIDAL DRUG DELIVERYSYSTEMS, vol. 66 (J. Kreuter ed., Marcel Dekker, Inc. (1994)).

IV. Lipids

In many embodiments, the MUC-1-based formulation may be lipidated, suchas with SEQ ID NO.: 2. As used herein, a “lipid” may be a myristyl,palmitoyl, or a lauryl molecule that can be attached to amino acids thatpossess functional oxygen, nitrogen, or sulfur groups. Such amino acidsinclude, but are not limited to, threonine, serine, lysine, arginine,and cysteine.

A “monolipopeptide” is a peptide to which only one lipid chain isattached. Similarly, a “dilipopeptide” is a peptide that has two lipidchains attached to either one or two amino acids. If the two lipidchains are attached to two amino acid residues, those residues can bespaced any number of amino acids apart. In cases where more than onelipid is attached, the lipids may either be the same lipid or may bedifferent lipids. Similarly, if more than two lipids are attached, twoor more of the lipids may be the same or all of the lipids may bedissimilar.

It is believed that a lipopeptide, such as BLP25, can be incorporatedinto a liposome because the lipid portion of that peptide spontaneouslyintegrates into the lipid bilayer of the liposome. Thus, in this case, alipopeptide may be presented on the “surface” of a liposome.Alternatively, a peptide may be encapsulated within a liposome.Techniques for preparing and formulating liposomes with molecules suchas peptides are well known.

V. Exemplary Adjuvants

The present MUC-1-based formulation may also include one or moreadjuvants. Alternatively, one or more adjuvants may be administeredeither before, in conjunction with, or after administration of theMUC-1-based formulation of the invention.

As is well appreciated, adjuvants are substances that act in conjunctionwith specific antigenic stimuli to enhance a specific response to anantigen. Monophosphoryl lipid A (MPLA), for example, is an effectiveadjuvant that causes increased presentation of liposomal antigen tospecific T Lymphocytes. Alving, C. R., Immunobiol., 187:430-446 (1993).MPLA may bind to toll-like receptors, which can lead to activation ofdefense signaling pathways that control the expression of various immuneresponse genes.

Lipid-based adjuvants, such as Lipid A and derivatives thereof, aresuitable for use with the MUC-1-based formulations. When incorporatedinto liposomes, a muramyl dipeptide (MDP) has also been shown toincrease adjuvancity (Gupta R K et al., Adjuvants-A balance betweentoxicity and adjuvancity,” Vaccine, 11, 293-306 (1993)).

Another class of adjuvants that may be used with the present inventionincludes stimulatory cytokines, such as interleukin-2 (IL-2). Thus, thepresent liposomal vaccines may be formulated with IL-2, or IL-2 may beadministered separately for optimal antigenic response. In manyembodiments, IL-2 is beneficially formulated with liposomes.

Synthetic mimics of adjuvants also may be co-formulated with use of theMUC-1-based formulations. For example, a lipid A mimic may be used inconjunction with the liposomal vaccine. One particular type of lipid Amimic is one in which one or both of the sugar units of the lipid Adisaccharide is replaced with at least the carbon skeleton ofpentaerythritol. See, for instance, WO 03/094850, which is incorporatedherein by reference.

VI. Exemplary Vaccine Formulations

When the MUC-1-based formulation is a vaccine, the vaccines may also beformulated with one or more pharmaceutically acceptable excipients. Theproperties of such excipients are well known in the art, but typicallyinclude excipients that are physiologically tolerable and inert orenhancing with respect to the vaccine properties of the inventivecompositions. Non-limiting examples of pharmaceutically acceptableexcipients include liquid vehicles such as sterile, physiologicalsaline. An excipient may be added at any point in formulating aliposomal vaccine or it may be admixed with the complete vaccinecomposition. One can easily determine both when to add the excipient andthe appropriate excipient for use with the vaccines of the invention.

One particular vaccine formulation may comprise about 300 μg of MUC-1lipopeptide BLP25 of SEQ ID NO: 2, about 150 μg of lipid A, and about 15mg of one or more additional liposomal lipids, such as dipalmitoylphosphatidiylcholine, cholesterol (DPMC), and dimyristoylphosphatidylglycerol (DPMG).

VII. Cyclophosphamide

Prior to treatment with a MUC-1-based formulation, a subject may be“pretreated” with cyclophosphamide. In many embodiments, the dose ofcyclophosphamide will be about 300 mg/m², about 400 mg/m², about 500mg/m², or about 600 mg/m². A dose of cyclophosphamide in the range ofabout 300 mg/m² is considered a low dose. In certain embodiments, thecyclophosphamide will be given in a single dose. In other embodiments,the cyclophosphamide will be given in more than one dose over a periodof time.

The use of a dose cyclophosphamide, such as 300 mg/m², may partlyovercome the immune suppression seen in some cancer patients. In variousanimal models, cyclophosphamide has been shown in certain subjects toaugment delayed-type hypersensitivity responses, increase antibodyproduction, abrogate tolerance, and potentiate antitumor immunity. Otherdrugs that affect the immune system in a similar manner tocyclophosphamide may also be used in pretreatment regimens with theformulations of the present invention.

VIII. Route of L-BLP25 Vaccine Administration and Targeting

The MUC-1-based formulations of the invention, including the vaccines,may be formulated for multiple routes of administration. Specific routesinclude any pharmaceutically suitable method of administration, such asvia intravenous, intramuscular, subcutaneous, or intradermal injection,aerosol, transdermal, pulmonary, nasal, oral, vaginal, rectal, ocular,local (powders, ointments or drops), buccal, intracisternal,intraperitoneal, or topical administration, and the like, or by acombination of these routes, administered at one time or in a pluralityof unit dosages. The vaccine or the liposomally-bound MUC-1 core repeatpeptide also may be administered via a formulation suitable fortransdermal delivery, such as via a transdermal patch.

Administration of vaccines is well known and ultimately depends upon theparticular formulation and the judgment of the attending physician.MUC-1 based formulations, such as L-BLP25 can be maintained as asuspension or they may be lyophilized and hydrated later to generate auseable formulation.

In some embodiments, such as the embodiment in example 1, one dose ofMUC-1-based formulation may be injected into several different sites.For example, in the embodiment of example 1, 1,000 μg of MUC-1-basedpolypeptide may be given in four sub-doses of approximately 250 μg each.In the case of injections, the amount of the injection is irrelevant aslong as the appropriate dose or sub-dose of inventive composition isbeing given. For example, one injection may be 1 cc (ml), while anotherinjection with the exact same dose may be 5 cc (ml). Furthermore, theamount in the sub-dose is meant as a non-limiting example only andembodiments where the sub-doses are more or less than ¼ of the full doseare anticipated.

The sub-dose or doses may be administered in the deltoid or tricepsregion of the upper arms, and the left and right anterolateral aspectsof the abdomen. However, these injection sites are meant as examplesonly. In some embodiments, only two sub-doses will be given and thesesub-doses may be given in any of the regions set forth above. In yetfurther embodiments, sub-doses or complete doses will be given incompletely different regions. If the MUC-1-based formulation isinjected, then an appropriate injection site can be easily determined.

To provide greater specificity, thus theoretically reducing the risk oftoxic or other unwanted effects during in vivo administration, in someembodiments inventive compositions will be targeted to the cells throughwhich they are designed to act, namely antigen-presenting cells. Thismay conveniently be accomplished using conventional targeting technologyto direct a liposome containing an immunogenic peptide to a particularlocation within the body. To target antigen presenting cells, forexample, mannose and the Fc portion of antibodies can be chemicallyconjugated to an antigenic peptide, or the targeting peptide may berecombinantly fused to the immunogenic lipopeptide. Other, similarstrategies will be familiar to the practitioner. Nonetheless, in someembodiments the inventive compositions will not be targeted to specificcell types or organs.

IX. Individuals for Treatment

Any subject diagnosed with a cancer susceptible to treatment with aMUC-1-based formulation, such as NSCLC and prostate cancer, may receivetreatment with the MUC-1-based formulations described herein.Alternatively, any individual who exhibits symptoms of any stage of acancer susceptible to treatment with a MUC-1-based formulation, such assymptoms of any stage NSCLC or any stage of prostate cancer, but who hasnot been formally diagnosed as having the cancer, may also receivetreatment with the MUC-1-based formulations. Furthermore, as statedabove, the MUC-1-based formulations may be given prophylacticly toprevent a subject from contracting a cancer susceptible to treatmentwith a MUC-1-based formulation, such as NSCLC or prostate cancer.

In selecting a subject with a cancer susceptible to treatment with aMUC-1-based formulation, such as a subject with NSCLC and/or prostatecancer, for treatment with the MUC-1-based formulation, it may bebeneficial to determine the level of MUC-1 in the subject's serum eitherbefore or during treatment. In certain cancer patients, high serum MUC-1levels have been correlated with poor prognosis. See, for instance, Pihlet al., Pathology, 12:439-447 (1980). Because an abnormal amount ofcirculating MUC-1 may inhibit or reduce the effectiveness of theinteractions of exogenous MUC-1-based formulations, knowing the amountof endogenous MUC-1 may assist in deciding the appropriate dose ofMUC-1-based formulations to be administered to a subject.

A. Tumor Marker CA27.29 (MUC1)

CA27.29 is the name for an antigen that is a particularly useful targetfor identifying MUC-1 protein, especially for identifying MUC-1 proteinfragments circulating in a subject's bloodstream, or in a suitablebiological sample, such as in a blood, urine, or serum sample.

The CA27.29 antigen can be detected by a monoclonal antibody that isspecific for the protein core of the MUC1 product. Minimum epitopemapping with overlapping synthetic peptides can be used to identify asuitable monoclonal antibody. One such antibody is B27.29.

Several variables, such as the number of MUC-1 tandem repeats and theposttranscriptional glycosylation of MUC-1 epitopes, can affect in vivodetection and quantification of MUC1-related markers. In vivo detectionof MUC-1 and its fragments may be further complicated by the presence ofcirculating anti-mucin autoantibodies, occasionally found in cancerpatients as a result of a host response to altered mucin biochemistry.These antibodies are capable of forming mucin-antibody mucinimmunocomplexes which may affect MUC1 detection, depending on the assayformat. See Gion et al., Clinical Chemistry, 45: 630-637 (1999), whichis incorporated herein by reference.

Any method can be used to detect or assay for amounts of CA27.29 antigenin a subject's blood sample, such as an enzyme-linked immunosorbentassay. Another assay is the ACS:180 BR assay (Bayer Diagnostics), whichis a fully automated competitive chemiluminescent immunoassay. A mousemonoclonal antibody, raised against a peptide epitope in the tandemregion of the MUC-1 backbone and labeled with acridinium ester, isincubated for 7.5 min with both the patient sample and purified CA27.29coupled covalently to paramagnetic particles (solid phase). Both theantigen in the sample and the solid-phase CA27.29 compete for binding tothe labeled antibody. Therefore, an inverse relationship is foundbetween the amount of antigen in the sample and the amount of relativelight units detected by the system. The assay can be performed accordingto the instructions of the manufacturer.

Another assay is the TRUQUANT BR radioimmunoassay, which uses monoclonalantibody B27.29 noted above to quantitate CA27.29 mucin antigen inserum. See MacLean et al., J. Immunother., 20(1):70-8 (1997).

Typically, in the present case exemplified in Example 4, a normal levelof CA27.29 antigen in serum or circulating blood is about 37.7 U/ml.Levels of serum MUC-1 that are greater than about 37.7 U/ml indicates anabnormal.

It is well within the purview of the skilled person to determinethreshold levels of circulating MUC-1 peptides, such as the CA27.29antigen, in different groups of individuals. That is, the value “37.7U/ml” is not necessarily a definitive threshold for all assayablepopulations. A “normal” quantity of circulating CA27.29, for instance,can be determined from a desired subpopulation and used as an indicatorto classify C27.29 quantities as either normal or abnormal.

In the present case, the upper limit for normal levels of circulatingMUC-1, as measured by C27.29 levels, was 37.7 U/ml. According to thepresent invention, therefore, an individual with a MUC-1 level that isabout 37.7 U/ml or lower than about 37.7 U/ml is a suitable candidatefor treatment with a MUC-1 based vaccine, such as with BLP25, or one ofthe other MUC-1 formulations described herein.

Also according to the present invention, a subject with a MUC-1 levelthat is at or lower than a different “threshold” normal level ofcirculating MUC-1 is also a suitable candidate for treatment with aMUC-1 based vaccine, such as with BLP25, or one of the other MUC-1formulations described herein.

B. HLA A2 Typing

The term HLA refers to the Human Leucocyte Antigen System, which iscontrolled by genes on the short arm of chromosome six. The HLA loci arepart of the genetic region known as the Major Histocompatibility Complex(MHC). The MHC has genes (including HLA) which are integral to normalfunction of the immune response. The essential role of the HLA antigenslies in the control of self-recognition and thus defence againstmicroorganisms. Some HLA antigens are recognised on all of the tissuesof the body, rather than just blood cells.

Human leucocyte antigen (HLA) A2 is the most heterogeneous allele at theHLA A locus, with approximately 56 different subtypes. Substantialheterogeneity in A02 distribution has been observed in populationsworldwide. HLA B40 is the most common allele associated with A2haplotypes. HLA A2 has also been implicated in a number of diseasesincluding Alzheimer's, psoriatic arthritis, vitiligo, and pulmonarytuberculosis.

It is possible to detect the presence of either the HLA A2 protein in anindividual's blood or tissue sample or of DNA or RNA. Hence, in thelatter kind of assay, the polymerase chain reaction (PCR) can beemployed to detect HLA A2 DNA, RNA, or cDNA. For instance, PCR-basedreverse blot sequence-specific oligonucleotide hybridisation technique.Alternatively Southern or Northern blots with an HLA A2-specific probemay be employed. On the other hand, an antibody can be used to detectHLA A2 protein.

The presence or absence of HLA nucleic acid or the HLA A2 protein is afactor to be considered in preparing the subject for treatment with aMUC-1 based vaccine, such as with BLP25.

X. Individuals with NSCLC

When subjects with NSCLC are to be treated with a MUC-1-basedformulation of the invention, subjects diagnosed as having stage IIIBlocoregional (LR), stage IIIB with malignant pleural effusion, or stageIV NSCLC specifically may be treated. Nevertheless, the presentinvention also encompasses the treatment of NSCLC individuals other thanthose having stage III locoregional, stage III pleural effusion, andstage IV disease. Thus, the present invention contemplates treatment ofstage IA, stage IB, stage IIA, stage IIB, stage IIIA, stage IIIB, stageIIIB locoregional, stage IIIB pleural effusion, and stage IVNSCLC-diagnosed patients. See Mountain C. F., Chest, 111(6):1710-7(1997), which is incorporated herein by reference.

A. Lung Cancer Staging

Generally, when the MUC-1-based formulations are used in subjects withNSCLC, the stage of NSCLC in the individual may be determined before,after, or during treatment. An outline of lung cancer staging is setforth below:

Normally in lung cancer, an increasing “stage” number correlates with aworse prognosis. To diagnose an individual at a particular stage, thesize and the location of the primary tumor (“T” value), as well as thedegree of nodal involvement and increasing probability of metastases(“N” value), are taken into consideration. Also noted when diagnosingindividuals is the absence (“M0”) or presence (“M1”) of metastases.

1. T Category

The T category is made up of subcategories, T1-T4, whereby an increasingnumber from 1 to 4 represents increasing size and local invasion by theprimary tumor. T1 and T2 are differentiated primarily on size, forexample, T1 is less than 3 cm while T2 is larger than 3 cm. T3 tumorstypically involve the chest wall, and include but are not limited to,the superior pulmonary sulcus, diaphragm, mediastinal pleura,pericardium, or proximal main stem bronchus, but may be resectable. T4tumors are not surgically resectable because they may have invaded themediastinum and may involve the heart, great vessels, trachea, carina,or esophagus, or in the case of a malignant pleural effusion, thepleura.

2. N Category

Nodal stages are divided into N1, N2, and N3. N1 nodes typically involveperibronchial or ipsilateral hilar nodes. These nodes are intrapleuralin position. N2 nodes typically involve ipsilateral mediastinal orsubcarinal nodes. N3 nodes typically involve contralateral hilar, ormediastinal, any scalene nodes, or supraclavicular nodes.

3. NSCLC Stages

The “stages” of NSCLC, therefore, represent distinct classifications ofNSCLC that are based on the various permutations of T, N, and M values.The recognized stages of NSCLC are as follows:

Occult Carcinoma:

In this category, patients are classified as TX N0 M0, meaning that theyhave had malignant cells detected in their bronchopulmonary secretions,but there is no tumor evident by bronchoscopic or radiographic methods.

Stage IA and Stage IB:

Stage IA is classified as T1 N0 M0 based upon a significantly better 5year survival outcome than patients with stage IB disease (T2 N0 M0).Surgery is the preferred treatment for these patients. In 1997, the 5year survival rate for patients surgically staged as stage IA was 67%and for stage IB was 57%.

Stage IIA and Stage IIB:

Stage IIA disease is defined as T1 N1 M0 and has a 55% survival rate at5 years based on surgical staging. Stage IIB disease is composed of T2N1 M0 and T3 N0 M0. The designation of T3 N0 M0 representsextrapulmonary extension of the tumor without lymph node involvement.The classification T3 N0 M0 is grouped with T2 N1 M0 because theirrespective 5 year survival rates for surgically staged disease, 38%versus 39%, are not significantly different. Surgery is also the primarytreatment for these individuals.

Stage IIIA:

Stage IIIA patients are considered to be resectable, while Stage IIIBpatients are not. Stage IIIA patients are defined by lesions withextrapulmonary extension (T3) and limited lymph node involvement (N1 orN2). The nodal involvement may extend to the ipsilateral mediastinal,and/or subcarinal lymph nodes. These patients are classified as eitherT3 N1 M0, or T1-3 N2 M0. As of 1997, the 5 year survival rate for stageIIIA disease was 23%.

Stage IIIB:

Stage IIIB classification refers to patients who have extrapulmonaryinvolvement including, but not limited to contralateral mediastinal orhilar lymph nodes; ipsilateral or contralateral supraclavicular orscalene nodes; extensive mediastinal nodes without distant metastases;or cytology positive malignant pleural effusion. These patients can beclassified as either T1-3 N3 M0 or T4 N0-3 M0. In 1997, the 5 yearsurvival rate for clinically staged disease was 5% with multimodaltherapy.

Stage IV:

Stage IV is defined by any metastatic involvement. These patients areclassified as M1 with any T and any N. As of 1997, more than a quarterof patients with NSCLC had clinical stage IV.

XI. Individuals with Prostate Cancer

Similar to the low survival rate of individuals with advanced stage lungcancer who under go multimodal therapy, men with prostate cancer whoexperience biochemical failure after a prostatectomy have fewtherapeutic options. One therapeutic option they do have is androgendeprivation therapy (ADT). Unfortunately, this therapy has significantmorbidity, especially if used for long periods of time.

In individuals with prostate cancer, it is known, for instance, thatprostate-specific antigen (“PSA”) levels in the blood tend to rise whenthe prostate gland enlarges. Accordingly, PSA is a good biological ortumor marker for prostate cancer. In individuals with more advanceddisease, treatment-induced decline in PSA correlates with improvedsurvival (Scher et al., J. Natl. Cancer Inst., 91(3):244-51 (1999)).

XII. Treatment of Individuals with NSCLC or Prostate Cancer

The present invention encompasses treatment with the MUC-1-basedformulations of the invention of NSCLC individuals in all NSCLC stagesas well as the treatment of individuals with prostate cancer, includingindividuals with prostate cancer that have PSA failure, post radicalprostatectomy. The use of the phrase “treating” is meant that theformulation or vaccine is useful for preventing, curing, reversing,attenuating, alleviating, minimizing, suppressing, or halting thedeleterious effects of a disease state, disease progression, diseasecausative agent, or other abnormal condition.

In some embodiments, individuals with either NSCLC or prostate cancermay have been previously treated with radiation or surgery, prior totreatment with the inventive compositions. Individuals may also undergotreatment with chemotherapy, radiation, or surgery before, while, orafter they have been treated with the MUC-1-based formulations of theinvention. In the case of these individuals, any accepted cancertreatment may be given before, during, or after treatment with theMUC-1-based formulations.

When choosing individuals to treat with the formulations and vaccines ofthe present invention, inclusion and exclusion criteria may be used. Forexample, in one embodiment, when NSCLC individuals are to be treatedwith the MUC-1-based formulation, individuals to be treated may be menor women over the age of 18 whose disease is stable or who haveresponded to treatment following completion of their first line standardchemotheraphy. Individuals other than those above may be treated withthe MUC-1-based formulations. In fact, some individuals treated with theinventive compositions will not have been treated with chemotheraphyprior to treatment with the MUC-1-based formulation.

XIII. Possible Inclusion and Exclusion Criteria for Individuals withNSCLC

In another embodiment, an individual with NSCLC chosen for treatment hasan Eastern Cooperative Oncology Group (ECOG) performance status of ≦2,with a neturophil count≧1.5×10⁹/L; platelet count≧100×10⁹/L;WBC≧2.5×10⁹/L and hemoglobin 90 g/L. Although the ECOG numbers may beused to evaluate individuals for treatment, particular ECOG numbers arenot required before, during, or after treatment.

Other inclusion criteria may include an expected survival of four monthsand where the individual has understood and signed a written consent. Ofcourse, these are not set inclusion criteria and treatment ofindividuals with lower life expectancies are envisioned. Furthermore, asMUC-1-based formulation become mainstream cancer treatments, individualswill likely have the inventive compositions prescribed and no signedwritten consent will be required.

Regarding individuals with NSCLC who may be excluded from treatment, theexclusion criteria are meant as guidelines only. In many cases,individuals exhibiting one or more of the exclusion criteria, includingall of the exclusion criteria, may still be treated with the MUC-1-basedformulations. Examples of exclusion criteria for NSCLC individualsinclude: (a) surgery or immunotherapy within four weeks prior totreatment, (b) immunosuppressive drugs including systemiccortiocosteriods within three weeks prior to treatment, (c) past orcurrent history of neoplasm other than lung carcinoma, (d) autoimmunedisease or recognized immunodeficiency disease, (e) clinicallysignificant hepatic or renal dysfunction, (f) significant cardiacdisease or active infection, or (g) individuals who had had asplenectomy.

XIV. Possible Inclusion and Exclusion Criteria for Individuals withProstate Cancer

Similar to individuals with NSCLC, individuals with prostate cancer mayalso be subjected to inclusion and exclusion criteria. Once again, thesecriteria are guidelines only and an individual with prostate cancer whodoes not satisfy any of the inclusion criteria or satisfies any of, orall of, the exclusion criteria may still be treated under the methods ofthe present invention. For individuals with prostate cancer, inclusioncriteria may include: (a) radical prostatectomy at least 6 months beforetreatment, (b) three consecutive increased serum PSA values post-radicalprostatectomy with at least a 50% increase above the post-prostatectomynadir, (c) no evidence of malignant disease at pre-treatment evaluationsas evidenced by negative pelvic CT and bone scan, (d) ECOG performancestatus of 0, 1, (e) normal haematological, hepatic and renal functiontests, (f) understand and sign a written informed consent; and (g)individuals who have ever been treated with hormonal therapy forprostate cancer (i.e., neoadjuvant treatment pre-RP) must have serumtestosterone within the normal range. As stated above, these inclusioncriteria are only guidelines and many individuals with differentcriteria may be treated using the methods of the invention. For example,individuals with prostate cancer who have not had a radicalprostatectomy may be treated. Furthermore, individuals who do not haveincreased serum PSA or whose serum PSA has not increased eitherconsecutively or is not above 50% as compared to the post-prostatectomynadir may also be treated.

Exclusion criteria that may be used, although not required, forindividuals with prostate cancer include: (a) hormonal therapy within 6months prior to treatment, (b) immunotherapy within 4 weeks prior totreatment, (c) radiotherapy to the prostate bed within one year prior totreatment, (d) treatment with immunosuppressive drugs such ascyclosporin or adrenocorticotropic hormone (ACTH) or requiring chronictreatment with corticosteroids, (e) known autoimmune or immunodeficiencydisease, or (f) clinically significant cardiac disease or activeinfection. Once again, these exclusion criteria are examples only. Forexample, individuals with both prostate cancer and clinicallysignificant cardiac disease may be treated with the methods of thepresent invention in individual cases.

XV. Effects of Treatment

Treatment with the MUC-1-based formulations described herein may resultin various effects. One effect of treating a NSCLC-diagnosed individual,specifically a stage IIIB NSCLC-diagnosed individual, with theMUC-1-based formulation is an increase in the length of survival.Similarly, administering the described MUC-1-based formulation anindividual may impact that individual's “quality of life” or“health-related quality of life.” An increase in survival, as well as animpact on quality of life, may also be seen in treated individuals withprostate cancer. Moreover, in certain individuals with prostate cancer,treatment with the MUC-1-based formulation will result in lower PSA,stabilized PSA, or decreased PSA doubling rates.

Comparisons of the effects of treatment with MUC-1-based formulationscan be made between treated individuals and individuals who are eitherundergoing no care or individuals who are undergoing best supportivecare (BSC). BSC comprises many alternative types of care that do notinclude treatment with the MUC-1-based formulation. For example, BSC,although usually discretionary depending on the circumstances, mayinclude psychosocial support, analgesics, and nutritional support. Insome embodiments, comparison of the effects of treatment will be madebetween individuals receiving differing amounts of the MUC-1-basedformulation. In yet further embodiments, individuals will undergo BSC inconjunction with treatment with the MUC-1-based formulations.

Before treatment of an individual with the MUC-1-based formulations ofthe present invention, individuals may undergo pre-treatment evaluation.A non-limiting example of a pre-treatment evaluation includes a completehistory and physical examination. The physical examination may includesuch things as a CT scan or X-ray. Individuals may also undergotreatment evaluations during the course of treatment. A treatmentevaluation may include monitoring the individual's vital signs,inspecting injection sites, and analyzing blood samples.

A treated individual may also be evaluated by determining the: (a) tumorsize, (b) tumor location, (c) nodal stage, (d) growth rate of the NSCLCor prostate cancer, (e) survival rate of the individual, (f) changes inthe individual's lung cancer or prostate cancer symptoms, (g) changes inthe individual's PSA concentration, (h) changes in the individual's PSAconcentration doubling rate, or (i) changes in the individual's qualityof life.

XVI. Increased Survival Time in NSCLC Individuals by AdministeringMUC-1-Based Formulation or BLP25 Liposome Vaccine

One of the advantages to treating an individual with NSCLC or prostatecancer with the MUC-1-based formulations of the invention is that theindividual may have a longer survival time than an individual who doesnot receive treatment with the inventive compositions. Survival ratesmay be determined by comparing the current number of survivors with thenumber of individuals who started treatment with the MUC-1-basedformulation. In other embodiments, survival rates may be compared topublished survival rates for a particular type of cancer. In general,the survival rate may be measured at any time following the start oftreatment.

For example, the survival rate may be measured at less than 6 monthsfollowing the start of treatment, greater than 6 months but less than ayear, a year or greater but less than 2 years, 2 years or greater butless than 5 years, or 5 or greater years. In some embodiments, anincreased survival rate will be evidence that the MUC-1-basedformulations of the invention are effecting a particular individual.

XVII. Maintaining the Quality of Life and Lung Cancer Symptoms byAdministering MUC-1-Based Formulations

As set forth above, another advantage of treating an individual withNSCLC or prostate cancer with the MUC-1-based formulations of theinvention is maintenance or an increase in the individual's quality oflife. Clinicians and regulatory agencies recognize that an individual's“quality of life” (“QoL”) is an important endpoint in cancer clinicaltrials. See, for instance, Plunkett et al., Clin. Lung Cancer,5(1):28-32 (2003), and Cella et al., J. Clin. Epidemiol., 55(3):285-95(2002), which are each incorporated herein by reference.

Four of the most important quality of life indicators are physical andoccupational function, psychologic state, social interaction, andsomatic sensations. In this respect, in individuals with NSCLC, two lungcancer questionnaires, the European Organization for Research andTreatment of Cancer (“EORTC”) and the Functional Assessment of CancerTherapy (“FACT-L”), can be used to assess an individual's, specificallyan individual's, health-related quality of life before, during, andafter treatment with the MUC-1-based formulations described herein.

It is anticipated that the methods of the invention may be used inconjunction with assessments according to various subscales that monitoran individual's Physical Well-being (PWB), Social/Family Well-being(SWB), Emotional Well-being (EWB), Functional Well-being (FWB), and LungCancer Symptom subscale (LCS). Although the Lung Cancer Symptom subscaleis obviously tailored to individuals with lung cancer, differentsubscales may be used with different types of cancer. Thus, a differentsubscale may be used with individuals with prostate cancer. Depending onwhich “Well-being” scores are combined, one may obtain a “FACT-L score”(the sum of all of the subscales) or a “Trial Outcome Score (TOI)” (thesum of the PWB, FWB, and LCS subscales). The TOI is a reliable indicatorof meaningful change in quality of life. See, Cella et al., supra.

The individual may be assessed for their FACT-L and TOI scores before,during, and after treatment with the MUC-1-based formulations of theinvention. For instance, the TOI score may be taken at baseline, i.e.,pre-treatment, and then at various intervals after treatment hasstarted, i.e., at 4 weeks, 8 weeks, 19 weeks, 31 weeks, or 43 weeks, orlonger. These various intervals are examples only and the quality oflife indicators may be taken at any appropriate time. For example, thefirst TOI score may be taken after the first treatment, instead of at abaseline. Then, the change in scores between various time points may becalculated to determine trends relating to improving, worsening, ormaintaining of quality of life.

It has been calculated that a decrease of 3 points or more from baselinefor LCS is a clinically meaningful worsening in lung cancer symptoms andan increase in 3 or more points is a clinically meaningful improvementin lung cancer symptoms. Likewise for TOI scores, a decrease of 7 ormore points indicates a worsening in quality of life, while an increaseof 7 or more points indicates an improvement in quality of life.

In some embodiments, a clinical improvement in lung cancer symptoms orquality of life will demonstrate that the MUC-1-based formulations areeffecting the particular individual.

Thus, administering the MUC-1-based formulations of the invention may beuseful in improving or maintaining the quality of life of treatedindividuals that have NSCLC or prostate cancer. In measuring the effecton the quality of life, an effect size can be determined from baselineor from any treatment point. In some embodiments, an effect size ofbetween 0.2 to <0.49 indicates a small effect, 0.5 to 0.79 indicates amoderate effect, and 0.8 or greater indicates a large effect. Thesenumbers are examples only and the effect size may change with treatmentof certain individuals.

Administration of the MUC-1-based formulations may also be useful inpreventing the worsening in quality of life seen over time in manycancer patients. For example, in some embodiments, administration of aMUC-1-based formulation such as the BLP25 liposomal vaccine may resultin quality of life indexes that essentially remain unchanged or do notreach the level of worsening or improving quality of life.

In one embodiment, the present invention encompasses improving ormaintaining the quality of life or improving or stabilizing lung cancersymptoms in an individual diagnosed with NSCLC by determining theindividual's TOI or LCS scores before, during, and after treatment withthe BLP25 MUC-1-based formulation described herein.

XVIII. Decreasing PSA Doubling Time

In some embodiments, treatment of individuals with prostate cancer withthe MUC-1-based formulations of the invention will result in a decreasein PSA concentrations, a stabilization of PSA concentrations, or adecrease in PSA doubling time. Generally, the effect of the MUC-1-basedformulations on PSA concentrations or PSA doubling time may be measuredat any time. For example, although PSA concentrations followingtreatment may be compared to a baseline value, the PSA concentration mayalso be compared between treatment points or between a specifictreatment point and the end of treatment. In certain embodiments, thePSA response will be confirmed during treatment.

XIX. Evaluation of Treatment Using Immune Function

In some embodiments, the response of individuals to MUC-1-basedformulations will be measured using tests of immune function, such as aT-cell proliferation response assays. In some embodiments, the resultsfrom T-cell proliferation response assays will be used to determinewhether the MUC-1-based formulation treatment is effecting anindividual. Results from these assays may also be used to determineindividual response to the formulations during different time pointsduring the course of the treatment.

Assays to measure proliferative T-cells are not particularly limitingand can be accomplished by any method known in the art. Comparison ofthe T-cell proliferation response may be undertaken to comparepre-treatment versus post-treatment response as well as to compareimmune responses within treatment.

XX. Other Cancers

The present invention also encompasses the treatment of other cancers inaddition to NSCLC and prostate cancer with the MUC-1-based formulationsdescribed herein.

Any individual who has a cancer that expresses MUC-1 may be targeted fortreatment with the MUC-1-based formulations. For instance, an individualwith a mucinous type cancer, or an adenocarcinoma that expresses a MUC-1protein may be targeted for treatment with the BLP25 liposome vaccine.Examples of adenocarcinomas include, but are not limited to ovariancancer, liver cancer, e.g., invasive cholangiocarcinomas of the liver,colon cancer, breast cancer, pancreatic cancer, e.g., invasive ductalcarcinomas of the pancreas, and kidney cancer, and multiple myeloma.Other cancers include cervical cancer, uterine cancer, and leukemia.Another cancer that expresses MUC-1 is head and neck cancer.

The examples below are intended to illustrate but not limit theinvention. While they are typical of how the methods of the presentinvention might be used, other methods, which conform to the spirit ofthe invention, are anticipated and may be used. Throughout thespecification, any and all references to a publicly available document,including a U.S. patent, are specifically incorporated by reference.

Example 1 Phase II Study of Liposomal MUC1 Vaccine for Treatment ofNSCLC

This example demonstrates the effects of a L-BLP25 vaccine on thetreatment of subjects with either stage IIIB locoregional or stage IVNSCLC.

Patients treated with the L-BLP25 vaccine demonstrated increasedsurvival rates. Furthermore, a clear advantage of the addition of BLP25liposome vaccine to best supportive care compared to best supportivecare alone was demonstrated by the maintenance in stable physicalwell-being throughout the treatment and maintenance phases of therapyand maintenance in the individuals' quality of life, as measured by theFACT-L total score and the Trial Outcome Index.

Methods:

The controlled, open-label Phase IIb trial enrolled 171 patients. Of the171 patients enrolled, 65 had IIIB locoregional disease. Of these, 35were randomized to treatment and 30 were randomized to best standardcare. The groups were well balanced in terms of age and ethnicity. Morefemale and ECOG 0 patients were randomized to treatment versus beststandard care (BSC) (51.4% and 36.7%, and 40.0% and 26.7%) and morepatients in the treatment arm received radiotherapy, in addition tochemotherapy, for cancer treatment prior to trial enrollment (91.4versus 76.7%).

The L-BLP25 vaccine used in this particular experiment was a lyophilizedpreparation consisting of (1) 1000 μg of a BLP25 lipopeptide, e.g., aMUC-1 peptide comprising SEQ ID NO: 2, (2) 500 μg immunoadjuvantmonosphoshoryl lipid A, and (3) three lipids: (i) 17.3 mg cholesterol,(ii) 3.6 mg dimyristoyl phosphatidylglycerol, and (iii) 29.1 mgdipalmitoyl phosphatidylcholine forming a liposomal product.

All patients in the L-BLP25 arm received at least five vaccinations,96.6% of these patients completed the primary phase and 69.3% continuedon to the maintenance phase of the treatment plan. Second-line therapywhile on study consisted mostly of chemotherapy (second or third-line),radiotherapy, and surgery. During the primary treatment period of thestudy, five patients on the L-BLP25 arm and 10 patients on the BSC armreceived second-line therapy. Of the patients who continued on to themaintenance period of the study, 43 patients on the L-BLP25 arm and 45patients on the BSC arm received second-line therapy.

To enhance the antigenic stimulation of a greater number of draininglymph nodes, the vaccine was administered to four anatomical sites. The1000 μg dose of L-BLP25 was given in four 0.5 mL subcutaneousinjections, with each injection containing one-fourth of the total dose.The sub-doses were administered in the deltoid or triceps region of theupper arms, and the left and right anterolateral aspects of the abdomen.

Generally, as used in this example, survival time is defined as the timefrom the date of randomization to the date of death. For patients aliveor lost to follow-up at time of analysis, the interval between date ofrandomization and date on which the patient was last known alive wascalculated and used as a censored observation in the analysis. In thisexample, survival was monitored at three-month intervals for 12 monthsafter completion of patient accrual.

A FACT-L QoL questionnaire was administered to all patients at specifictime points. The QoL analysis included evaluation of mean FACT-Lindividual change scores from baseline to week four and week eight,graphic representation of QoL scores over time, and area under the curveanalysis for total and subscale scores. The effect size of the qualityof life changes between treatment arms was determined from baseline. Aneffect size between 0.2 to <0.49 indicates a small effect, 0.5 to 0.79indicates a moderate effect, and 0.8 or greater indicates a largeeffect.

Results:

As shown in FIG. 2, the observed two-year survival for stage IIIBlocoregional patients is 60% for the vaccine arm versus 36.7% for thecontrol arm, demonstrating a significant increase in life expectancy of23.3%. In the overall patient population, the two-year survival is 43.2%for the vaccine arm versus 28.9% for the control arm, demonstrating anincrease in life expectancy of 14.3%. See FIG. 1. The median survival ofstage IIIB locoregional patients undergoing only best standard care wassimilar to the overall median survival of the entire group undergoingbest standard care at 13.3 months. In contrast, the overall mediansurvival for the stage IIIB locoregional patients undergoing treatmentwith L-BLP25 has remained unmet with a minimum median of 24 months,demonstrating an increase in life expectancy of at least 10.7 months.This is surprising and unexpected, as prior to the introduction of theMUC-1 compositions of the invention, no viable treatment options forthis category of patients could produce such results.

Regarding the quality of life, a clear advantage for the L-BLP25 armcompared to the BSC arm was demonstrated. More patients in the L-BLP25arm showed either a clinically meaningful improvement or did not changecompared to patients in the BSC arm. In the BSC only arm, more patientsdemonstrated a clinically meaningful worsening in the Trial OutcomeIndex (TOI).

Method:

Stage IIIB locoregional (LR) disease and stage IIIB with malignantpleural effusion (PE)/IV subgroup comparison analysis between treatmentand BSC only patients was performed for the FACT-L total score, thevarious subscales, and TOI using a T-test. A negative Total/TOI changescore indicates a worsening in QoL, whereas a positive Total/TOI changescore indicates an improvement. The subgroup analysis indicates a betterQoL for stage IIIB LR patients treated with L-BLP25. This is consistentwith previous data demonstrating a clinically meaningful improvement insurvival in stage IIIB LR patients treated with BLP25 (p=0.0692).

The results of the Quality of Life comparisons are shown in Table 1below:

TABLE 1 FACT-L Quality of Life Comparison in NSCLC Study IIIBLRIIIBPE/IV QoL IIIBLR TX BSC P IIIBPE/TV TX BSC P FACT-L Total Score ΔFrom Baseline 0.6 ± 12.1  −7.5 ± 12.7 .027 −0.2 ± 13.2 −8.6 ± 22.2 .072week 19 Δ From Baseline 2.9 ± 14.2 −8.0 ± 9.0 .008 −2.4 ± 10.3 −0.7 ±17.0 .737 week 31 TOI Δ From Baseline 0.5 ± 8.2   −6.5 ± 10.9 .014 −1.0± 10.6 −6.6 ± 15.0 .110 week 19 Δ From Baseline 1.2 ± 10.2 −6.5 ± 8.3.016 −1.3 ± 8.7  −2.4 ± 10.5 .761 week 31Study Design

Week −2: Administration of a FACT-L QoL questionnaire.

Week −2: Patients were randomized to either L-BLP25 plus best standardof care or to best standard of care alone (best standard of careincludes palliative radiotherapy and/or second line chemotherapyaccording to current standard clinical practice and may also includepsychosocial support, analgesics and nutritional support).

Week −2: Pretreatment evaluation (complete history, physicalexamination, and clinical laboratory studies). Evaluations of otherpotential disease sites were conducted, if clinically warranted, to ruleout progressive disease in other areas. Women of childbearing potentialwere required to have a negative pregnancy (HCG) test before treatment.

Day −3: Treatment arm patients received a single intravenous dose of 300mg/m² cyclophosphamide.

Weeks 0 to 7: L-BLP25 vaccinations #1 to #8 (primary treatment period).Patients on the L-BLP25 arm had vital signs assessed and previousinjection sites inspected prior to each L-BLP25 treatment. Vital signswere also monitored one-hour following each L-BLP25 treatment. Patientswere given diary cards following each vaccination to record any adverseevents and previous injection sites were evaluated at each subsequentvisit. Toxicity was graded according to the CALGB Expanded Criteria.

Week 4: Treatment evaluation and safety and immunology blood work forpatients in the treatment arm. FACT-L QoL Questionaire to all patients.

Week 8: Treatment evaluation (physical examination, ECOG status, vitalsigns, treatment site inspection for the L-BLP25 arm, and adverse eventsassessment). Blood samples were also drawn and analyzed for standardsafety (hematology and chemistry) as well as immune response. FACT-L QoLQuestionaire to all patients.

Week 19+: Maintenance vaccinations (6 week intervals) and treatmentevaluations (12 week intervals). Patients on the L-BLP25 arm hadtreatment evaluations and safety blood work performed at eachmaintenance vaccination as well as an immunology profile examination oneweek following the first maintenance vaccination. FACT-L QoLQuestionaire to all patients.

Patient Population

Inclusion Criteria

1. Men and women over the age of 18 with NSCLC whose disease was stableor who had responded to treatment following completion of their firstline standard chemotherapy.

2. Eastern Cooperative Oncology Group (ECOG) performance status of ≦2,with a neturophil count≧1.5×10⁹/L; platelet count≧100×10⁹/L;WBC≧2.5×10⁹/L and hemoglobin 90 g/L.

3. Expected survival of four months.

4. Understood and signed a written consent.

Exclusion Criteria

1. Surgery or immunotherapy within fours weeks prior to study entry.

2. Immunosuppressive drugs including systemic corticosteroids withinthree weeks prior to study entry.

3. Past or current history of neoplasm other than lung carcinoma.

4. Autoimmune disease or recognized immunodeficiency disease.

6. Clinical significant hepatic or renal dysfunction.

7. Significant cardiac disease or active infection, or patients who hadhad a splenectomy.

TABLE 2 Patient Characteristics of NSCLC Study L-BLP25 + BSC BSC Total N= 88 N = 83 N = 171 Age at Randomization: (years) Median 59.5 59 59Gender: N (%) Female 36 (40.9) 40 (48.2) 76 (44.4) Male 52 (59.1) 43(51.8) 95 (55.6) ECOG Performance Status: N (%) 0 31 (35.2) 22 (26.5) 53(31.0) 1 53 (60.2) 57 (68.7) 110 (64.3)  2 4 (4.5) 4 (4.8) 8 (4.7)Disease stage: N (%) IIIB LR 35 (39.8) 30 (36.1) 65 (38)  IIIB MPE or IV53 (60.2) 53 (63.9) 106 (62)   Response to First-line Therapy: N (%)Stable Disease 39 (44.3) 38 (45.8) 77 (45.0) Clinical Response (PR orCR) 49 (55.7) 45 (54.2) 94 (55.0)

Example 2 T-Cell Proliferation Response Assays

This example demonstrates that the MUC-1 formulations of the inventionwere directly responsible for the increase in median survival shown inExample 1.

Lymphoproliferation assays were performed using the patients enrolled inthe study of Example 1 to monitor MUC1 antigen specific TH response(proliferation of helper T-cells) prior to and following vaccinations tomeasure the dynamics of the patient's anti-MUC1 cellular immuneresponse. T-cell proliferation assays were performed on patients in theL-BLP25 arm both pre-immunization and at several time points postimmunization.

Of the patients in the L-BLP25 arm, 78 were evaluated for a T-cellproliferative response. Sixteen patients were determined to have apositive MUC1 specific T-cell proliferative response that was induced bythe L-BLP25 vaccine (the response did not exist pre-immunization). Ofthe sixteen patients who developed an immune response, two had stageIIIB locoregional disease, with the remaining patients having stage IVdisease. The median survival of the patients on the L-BLP arm with apositive proliferative response was 27.6 months while those patientswith a negative proliferative response had a median survival of 16.7months. These results demonstrate that the MUC-1 formulation of theinvention was directly responsible for the increase in median survivalof life expectancy of 10.9 months.

Example 3 Phase II Study of Liposomal MUC1 Vaccine in PSA FailuresPost-Radical Prostatectomy (RP)

This example shows the immunotherapeutic effects of L-BLP25 vaccine onthe PSA levels in men with rising PSA following radical prostatectomy.

At the end of the primary treatment period (week 8), 8/16 patients hadstable PSA. One patient maintained stable PSA through to the end of thestudy period (week 49). There was a noted prolongation in PSA doublingtime (“PSADT”) for all but one patient enrolled. The doubling time isthe length of time it takes for an individual's PSA level to double andis a factor used to predict survival following surgery in individualswith prostate cancer. The present data show that, in 6/16 patients, thedoubling time exceeded 50%.

Methods:

Men with biochemical failure as evidenced by 3 rises in PSApost-prostatectomy were enrolled. This included sixteen patients, with amedian age of 60, an ECOG score of 0 or 1, and median Gleason score of7. Primary endpoints were efficacy (as measured by PSA response) andsafety of a MUC1 liposomal vaccine (L-BLP25). Changes in PSA doublingtime (PSADT) were also evaluated. Patients received a single intravenousdose of 300 mg/m² cyclophosphamide (CTX) followed by 8 weeklysubcutaneous vaccinations with L-BLP25 containing 1,000 μg antigen(treatment). Subsequent vaccinations were given at 6-week intervalsthrough week 49 (maintenance). PSA concentrations were measured duringthe treatment and maintenance phases and PSADT was calculated for theseintervals and compared to PSADT prior to enrolment.

All 16 patients received CTX and 15/16 completed the treatment period.Ten patients completed the maintenance period. The most common adverseevents following treatment were nausea (31%) and fatigue (25%); however,none of these adverse effects were worse than grade 1.

Results:

After induction, 8/15 evaluable patients had either stabilization ordecrease in PSA (as per PSA Working Group definition). At the laston-study PSA measurement, one patient maintained a stable PSA. 6/15patients had a >50% prolongation of PSADT compared to pre-study PSADT.

Primary endpoint evaluation of PSA stabilization or reduction in thisindividual population by the use of L-BLP25 vaccine was as follows:

-   -   8/16 individuals had PSA stability after primary treatment        period;    -   1/16 individuals retained PSA stability by the end of the        maintenance period; and    -   PSADT was prolonged in 14/15 subjects by use of vaccine; 6/16        individuals had prolongation of PSADT by >50%.

Study Design:

Week −2: Pre-treatment evaluation (physical exam, PSA concentrationmeasurement, pelvic CT, and bone scan).

Day −3: Cyclophosphamide pretreatment.

Weeks 0 to 7: L-BLP25 Vaccinations #1 to #8 (primary treatment period).

Week 8: Primary treatment period evaluation including PSA response.

Week 13: Confirmation of PSA response.

Weeks 13, 19, 25, 31, 37, 43 & 49: L-BLP25 Vaccinations #9 to #15(maintenance period).

Week 43: Evaluation of PSA response.

Week 49: Confirmation of PSA response.

Week 50: Maintenance treatment evaluation.

Individual Population:

Inclusion Criteria

1. Radical prostatectomy at least 6 months prior to study entry.

2. Three consecutive increases in serum PSA values post-radicalprostatectomy with at least a 50% increase above the post-prostatectomynadir.

3. No evidence of malignant disease at pre-treatment evaluations asevidenced by negative pelvic CT and bone scan.

4. ECOG performance status of 0, 1.

5. Normal haematological, hepatic and renal function tests.

6. Understood and signed a written informed consent.

7. Serum testosterone within the normal range for all patients who haveever been treated with hormonal therapy for prostate cancer (i.e.neoadjuvant treatment pre-RP).

Exclusion Criteria

1. Hormonal therapy within 6 months prior to study entry.

2. Immunotherapy within 4 weeks prior to study entry.

3. Radiotherapy to the prostate bed within one year prior to studyentry.

4. Treatment with immunosuppressive drugs such as cyclosporin oradrenocorticotropic hormone (ACTH) or required chronic treatment withcorticosteroids.

5. Known autoimmune or immunodeficiency disease.

6. Clinically significant cardiac disease or active infection.

TABLE 3 Patient Characteristics for Prostate Cancer Study Age (yrs): n16   Mean ± S.D. 60.4 ± 7.7  Median 60.0  25%/75% 54.5/66.0 Range 46.0to 74.0 ECOG Performance Status: n (%)  0 13 (81%)  1 3 (19%) GleasonGrade:  6 3 (19%)  7 10 (63%)  8 3 (19%) Initial Diagnosis to StudyEntry (years): Mean ± S.D. 3.8 ± 2.5 Median 3.2 Range 1.0 to 9.5Post-prostatectomy Nadir to Study Entry (years): Mean ± S.D. 3.1 ± 2.3Median 2.8 Range 0.6 to 9.1 Baseline PSA μg/L: Mean 3.8 Median 0.425%/75% 0.1/0.8 Treatment Received Total Number Receiving Treatment n(%) Cyclophosphamide 16 (100.0) Primary Treatment Period Vaccinations  116 (100.0)  2 16 (100.0)  3 16 (100.0)  4 16 (100.0)  5 16 (100.0)  6 16(100.0)  7 15 (93.8)  8 15 (93.8) Maintenance Treatment PeriodVaccinations  9 14 (87.5) 10 14 (87.5) 11 13 (81.3) 12 12 (75.0) 13 11(68.8) 14 10 (62.5) 15 10 (62.5)

TABLE 4 PSA VALUES Change in PSA from Baseline to Week 8 (primarytreatment period) Per Patient Subject Baseline PSA Week 8 PSA Responseat Number (μg/L) (μg/L) Week 8 001 20.00 Did not reach week 8 NotAssessed 002 35.00 48.00 Progression 003 0.07 0.07 Stable PSA 004 0.470.46 Stable PSA 005 0.17 0.14 Stable PSA 006 0.89 0.94 Stable PSA 0070.36 0.45 Progression 008 0.58 0.79 Progression 009 0.10 0.11 Stable PSA010 0.08 0.10 Progression 011 0.59 0.82 Progression 012 0.11 0.18Progression 013 0.13 0.18 Progression 014 0.71 0.64 Stable PSA 015 1.801.90 Stable PSA 016 0.38 0.34 Stable PSAAdverse Events

Total Number of Patients with Adverse Events, n (%) 14 (87.5)

Patients Reporting Adverse Events with 10% or Greater Frequency

Nausea 5 (31.3) Fatigue 4 (25.0) Diarrhoea NOS (not otherwise specified)3 (18.8) Influenza-like illness 3 (18.8) Nasopharyngitis 3 (18.8)Constipation 2 (12.5) Headache NOS 2 (12.5) Pain NOS 2 (12.5)

Six of sixteen patients reported no injection site reactions of anytype. Nine patients reported erythema. No ulcerations occurred. Noevents occurred that were severe or precluded further vaccinations.

TABLE 5 PSA DOUBLING TIME Interval A Interval B PSADT Difference PSADTPSADT Between Interval A & Patient ID (days) (days) Interval B 002 173476 175%  003 133 291 119%  004 345 393 14% 005 302 342 13% 006 172 185 8% 007 309 347 12% 008 173 332 92% 009 404 637 58% 010 508 595 17% 011165 257 56% 012 241 97 −60%  013 84 112 33% 014 479 844 76% 015 227 28827% 016 344 385 12% Mean 271 372 44% Interval A = Prestudy (from thefirst of 3 consecutive rising prestudy PSA's prior to study entry tobaseline) Interval B = Maintenance (from Week 8 to end of study)

Example 4 Tumor Marker CA27.29

FIG. 1 lists the frequency distribution of patient CA27.29 values byvisit, arm (BLP25 or Control), and normal/abnormal level. Determinationof serum levels of MUC1 (CA27.29) was performed on baseline samples of166 patients (there was no data generated for 4 patients on BLP25 arm(3—no sample received; 1—improper labelling) and 1 patient on controlarm (1—data not received for entry) and on post 8 immunization samplesof 153 patients. No significant differences are seen between the arms bythe CA27.29 levels.

FIGS. 5-9 display the Kaplan-Meier Curves of survival duration byCA27.29 (MUC1). FIG. 5 displays the survival curve for patients withnormal levels of the antigen at baseline compared to those patients whohad abnormal levels. The upper limit of normal for serum levels of MUC1as determined in this assay (Truquant® BR Radioimmunoassay, based onsera from 1004 healthy female donors) is 37.7 U/ml. Levels of serum MUC1that were greater than 37.7 U/ml were considered abnormal. There were124 patients at baseline with normal levels of CA27.29 (63 in the BLP25arm and 61 in the control arm) and 42 with abnormal levels (21 in theBLP25 arm and 21 in the control arm). The median survival of thosepatients having normal levels of CA27.29 at baseline was 19.5 monthswhile the median survival of those having abnormal levels was 10.5months (Cox p<0.0001).

FIGS. 6 and 7 illustrate the survival of patients in each arm based uponwhether they have normal or abnormal CA27.29 levels at baseline. In theBLP25 arm (FIG. 6), median survival of those patients with normal levelsof CA27.29 was 24.2 months while that of patients with abnormal levelswas 9.8 months (Cox p=0.0006). In the control arm (FIG. 7), the mediansurvival of patients with normal levels of CA27.29 was 15.1 months ascompared to 11.3 months for those with abnormal CA27.29 levels (Coxp=0.0042).

FIGS. 8 and 9 show the differences in survival of patients in Arm A(BLP25) versus those in Arm B (Control) based on whether they havenormal or abnormal pre-immunization CA27.29 serum levels. As illustratedin FIG. 8, patients with pre-existing normal levels of CA27.29 in theBLP25 arm had a median survival of 24.2 months as compared to thosepatients in the Control arm who had a median survival of 15.1 months(Cox p=0.0605). In FIG. 9, the patients with pre-existing abnormallevels of CA27.29 in the BLP25 arm had a median survival of 9.8 monthsand those patients on the control arm had a median survival of 11.3months (Cox p=0.5234).

FIG. 10 shows the Kaplan-Meier Curves of survival duration by T cellproliferation. T-cell proliferation assays were performed on patients inArm A (BLP25) both pre-immunization and at several time points postimmunization. These patients were then listed as having a positive ornegative response if one or more of the time points showed a responsethat was different than the response at the pre-immunization time point.Of the patients on the BLP25 arm, 78 were evaluated for a T cellproliferative response. Sixteen patients where determined to have apositive MUCI specific T cell proliferative response that was induced bythe L-BLP25 vaccine. The Kaplan Meier Survival Curve (FIG. 10) showsthree patient populations: the control arm, the positive proliferatorson the BLP25 arm and the negative proliferators on the BLP25 arm. Themedian survival of those patients on the BLP25 arm with a positiveproliferative response was 27.6 months while those patients with anegative proliferative response had a median survival of 16.7 months;and patients on the control arm had a median survival of 13 months (Coxp=0.2148).

Example 5 HLA Typing

HLA typing was performed on all patients in Arm A (BLP25) and in Arm B(control). This typing was done to consider the possibility that the HLAClass I and II alleles that a patient expresses may be of importance tothe efficacy of the vaccine. HLA typing was performed by DNA analysisnot serology and the nomenclature used is that for DNA. For each arm,the % frequency and the n for a given allele were determined. Forsurvival analysis, HLA alleles that had an n>_(—) 19 in either arm werechosen. Additionally the analysis was performed for HLA 11 as there is aknown MUCI CTL epitope for this haplotype.

FIG. 11 illustrates the survival analyses that were performed for thechosen HLA types between treatment arms. The table lists thehaplotype/allele, the total number of patients with that haplotype (eachpatient has more than one haplotype), the number of patients with thattype on the BLP25 and Control arms, the median survival for patientswith those haplotypes in the BLP25 and Control arms and the statisticalsignificance by Log-Rank test and Cox H. R. analyses when comparing thetreatment.

FIGS. 12-16 illustrate the results of the Kaplan-Meier Survival analysesfor a few of the HLA alleles. These analyses were corrected for responsestratification and staging at study entrance in the Cox H. R. analysis.FIG. 12 shows the survival of patients with HLA A02 in each of the studyarms. There were 44 patients with this type in the BLP25 arm and 36patients in the Control arm. HLA A02 patients on BLP25 arm had a mediansurvival of 22 months compared to a median survival of 11.9 months forthe HLA A02 patients on the control arm (Cox p=0.0063). FIG. 13demonstrates the differences in survival between the two arms inpatients with the DQB 1-05 allele. The median survival of patients withthis allele in the BLP25 arm (n=25) has not yet been reached whereas themedian survival of those DQB1-05 patients in the control arm (n=27) is12.9 months (Cox p=0.0213). FIG. 14 displays the survival of patients inboth arms who have the DRB1-04 haplotype. There were 22 patients in theBLP25 arm with this haplotype and 23 patients on the control arm. Mediansurvival was 22 months for those patients on the BLP25 arm and 11.8months for those patients on the control arm (Cox p=0.0043).

FIG. 15 shows another survival curve for patients in both arms who havethe DQB 1-02 allele. Patients with this allele in the BLP25 arm (n=31)had a median survival of only 9.8 months while those in the control armhad a median survival of 16 months (Cox p=0.0424).

FIG. 16 illustrates additional survival analyses within the treatmentarms for the same haplotypes listed in FIG. 17. The first half of thetable lists the haplotype, the number of patients in the BLP25 arm thatare positive and negative for the haplotype, The Log-Rank and Cox H. R.p values, and the median survivals of the patients in the BLP25 arm whoare positive and negative for that haplotype. The second half of thetable gives the same information for those patients on the control arm.

The last Kaplan-Meier Survival curve (FIG. 18) displays patients in theBLP25 arm only who have the CW07 allele (n=45) versus those patients whodo not have the allele (n=43). The median survival of the patients withthe CW07 allele on the BLP25 arm had a median survival of only 12.6months while those on the arm with out the allele had a median survivalof 24.2 months (Cox p=0.0171). There was no real difference in thesurvival of patients on the control arm with and without the CW07 allele(Graph not shown). In addition, there was no real difference in survivalof patients in the BLP25 arm who had the CW07 allele and those on thecontrol arm who had the allele (data not shown).

As will be understood, all ranges disclosed herein also encompass anyand all possible subranges and combinations of subranges thereof. Thus,any listed range can be easily recognized as sufficiently describing andenabling the same range being broken down into at least equal halves,thirds, quarters, fifths, tenths, etc. As a non-limiting example, eachrange discussed herein can be readily broken down into a lower third,middle third and upper third, etc. As will also be understood, alllanguage such as “up to,” “at least,” “greater than,” “less than,” “morethan” and the like include the number recited and refer to ranges whichcan be subsequently broken down into subranges as discussed above. Inthe same manner, all ratios disclosed herein also include all subratiosfalling within the broader ratio.

Also, where members are grouped together in a common manner, such as ina Markush group, the present invention encompasses not only the entiregroup listed as a whole, but each member of the group individually andall possible subgroups of the main group. Accordingly, for all purposes,the present invention encompasses not only the main group, but also themain group absent one or more of the group members. The presentinvention also envisages the explicit exclusion of one or more of any ofthe group members in the claimed invention.

All references, patents and publications disclosed herein arespecifically incorporated by reference thereto. Unless otherwisespecified, “a” or “an” means “one or more”.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

What is claimed is:
 1. A method for treating a subject with prostatecancer comprising: (a) selecting for treatment a subject having prostatecancer wherein the subject has been HLA-typed and does not have an HLAhaplotype selected from DQB1-02 and CWO7, and (b) administering to thatsubject, for a period of time, a MUC-1-based formulation, wherein theformulation comprises: (i) a liposome; and (ii) at least one polypeptidecomprising the amino acid sequence selected from the group consisting ofthe amino acid sequence of SEQ ID NO. 1 and the amino acid sequence ofSEQ ID NO.
 2. 2. The method of claim 1, wherein the formulation furthercomprises at least one adjuvant.
 3. The method of claim 2, wherein theadjuvant is selected from the group consisting of lipid A, muramyldipeptide, alum, a cytokine, and a combination thereof.
 4. The method ofclaim 3, wherein the lipid A is monophosphoryl lipid A or a syntheticlipid A.
 5. The method of claim 3, wherein the cytokine isinterleukin-2.
 6. The method of claim 1, further comprising a step (c)evaluating the treated subject.
 7. The method of claim 6, whereinevaluating the treated subject comprises measuring an immune reaction inthe treated subject.
 8. The method of claim 7, wherein measuring theimmune reaction in the treated subject comprises measuring a T-cellproliferation.
 9. The method of claim 6, wherein evaluating the treatedsubject comprises determining at least one of: (a) tumor size, (b) tumorlocation, (c) nodal stage, (d) growth rate of the prostate cancer, (e)survival rate of the subject, (f) changes in the subject's lung canceror prostate cancer symptoms, (g) changes in the subject's PSAconcentration, (h) changes in the subject's PSA concentration doublingrate, or (i) changes in the subject's quality of life.
 10. The method ofclaim 1, wherein the formulation is a BLP25 liposome vaccine comprising:(c) a MUC-1 peptide comprising the sequence of SEQ ID NOS: 1 or 2; (d)one or more adjuvants, and (e) one or more additional liposomal lipids.11. The method of claim 10, wherein the BLP25 liposome vaccine isprovided in a kit.
 12. The method of claim 1, wherein the step ofadministering is by injection, aerosol, nasal, vaginal, rectal, buccal,occular, local, topical, intracisternal, intraperitoneal, or oraldelivery, and wherein the injection is an intramuscular, intravenous,subcutaneous, intranodal, intratumoral, intraperitoneal, or intradermalinjection.
 13. The method of claim 1, wherein the period of time isselected from the group consisting of at least about 2 weeks, at leastabout 4 weeks, at least about 8 weeks, at least about 16 weeks, at leastabout 17 weeks, at least about 18 weeks, at least about 19 weeks, atleast about 20 weeks, at least about 24 weeks, at least about 28 weeks,at least about 32 weeks, at least about 36 weeks, at least about 40weeks, at least about 44 weeks, at least about 48 weeks, at least about52 weeks, at least about 60 weeks, at least about 68 weeks, at leastabout 72 weeks, at least about 80 weeks, at least about 88 weeks, atleast about 96 weeks, and at least about 104 weeks.
 14. The method ofclaim 1, wherein the individual is treated with cyclophosphamide priorto (b).
 15. The method of claim 1, further comprising calculating acombined score of the subject's physical well-being, functionalwell-being, and lung cancer or prostate cancer symptoms before, during,and after the period of time wherein the subject had been diagnosed withprostate cancer.
 16. The method of claim 1, wherein the period of timeis at least about 6 months, at least about 12 months, at least about 18months, at least about 24 months, or longer than 24 months.
 17. Themethod of claim 10, wherein the amount of MUC-1 peptide is about 300 μg.18. The method of claim 10, wherein the adjuvant is lipid A.
 19. Themethod of claim 18, wherein the amount of lipid A is about 150 μg. 20.The method of claim 10, wherein the amount of additional liposomallipids is about 15 mg.
 21. The method of claim 10, wherein the MUC-1peptide comprises the sequence depicted in SEQ ID NO:
 1. 22. The methodof claim 10, wherein the MUC-1 peptide comprises the sequence depictedin SEQ ID NO:
 2. 23. The method of claim 21, wherein the MUC-1 peptideis lipidated.
 24. The method of claim 1, wherein the subject has risingPSA post-radical prostatectomy.